AbstractKinase‐catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase‐catalyzed biotinylation, where ATP‐biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase‐catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag to phosphatases was characterized. We found that the phosphorylbiotin group on peptide and protein substrates was relatively insensitive to protein phosphatases. To understand how phosphatase stability would impact phosphoproteomics research applications, kinase‐catalyzed biotinylation of cell lysates was performed in the presence of kinase or phosphatase inhibitors. We found that biotinylation with ATP‐biotin was sensitive to inhibitors, although with variable effects compared to ATP phosphorylation. The results suggest that kinase‐catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low‐abundance phosphoproteins or characterizing the influence of inhibitor drugs on protein phosphorylation.